The Zn(AC)₂⋅2H₂O and Na₂S⋅9H₂O were obtained from Sigma–Aldrich. Thioctic acid (TA), 3-Mercaptopropionic acid (MPA), N-Hydroxysulfosuccinimide sodium salt (NHSS), and Eu(NO)₃⋅6H₂O were purchased from Aladdin Reagent Company Limited (Co., LTD). 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), Bovine serum albumin (BSA), goat anti-human immunoglobulin (IgG) and human IgG were purchased from Beijing Dingguo biotechnology Co., LTD. All chemicals were of analytical grade in purity and directly used without being purified further.

Aqueous synthesis not only solves the problem of the water solubility of the nanomaterials, but also is simpler, less toxic and less expensive than the metal–organic chemical method. Thus, ZnS:Eu nanomaterials were prepared by a modified aqueous method.^[13] The improvement was that the carboxyl groups were introduced by MPA to gain a biofunctional surface. The detailed process is as follows: 300-μl MPA was added into 50-ml 75-mM Zn(AC)₂⋅2H₂O aqueous solution; 50-ml 0.75-mM Eu(NO)₃⋅6H₂O was added in the mixing process for the 1% doped ZnS:Eu sample. After mixing for 20 min, 50-ml 75-mM Na₂S⋅9H₂O solution was slowly added to the mixed solution to provide element sulphur. Stirring continued for 3 h to obtain ZnS:Eu sample. The concentration of the Eu³⁺ was amplified to prepare 2% (3%, 4%, and 5%) doped samples.

The principle for preparing the fluorescent probe and the sandwich immunoassay is depicted in Fig. 1. For preparing the probe, the as-prepared ZnS:Eu nanomaterial solution (2 ml) was dispersed in deionized water (0.5 ml) after centrifugation. An EDC/NHSS aqueous solution (0.1 M/0.02 M) was mixed with the ZnS:Eu solution and was rocked for 15 min, to assist the coupling of the antibody.^[14] Then, 660-nM goat anti-human IgG was added and rocked at 37 °C for 1 hour. The 1.5-ml BSA solution with a mass concentration of 1% was used to block the remaining bind sites to avoid nonspecific absorption. Finally, the obtained probe was dispersed in aqueous solution (1.5 ml) after being centrifuged.

Fig. 1.

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	Fig. 1. (color online) Schematic diagram of the ZnS:Eu based immunoassay.

The Au substrate assembly we used in the present investigation (for details, see the research work of Meyerhoff et al.)^[15] was obtained by depositing a layer of Au on a single-crystal silicon in vacuum. Because Au and other sulfur containing groups are prone to coordination processes,^[16] carboxyl groups were introduced by immersing the Au substrate in a 2% ethanol solution of TA for 24 hours. In each of the following steps, the substrates were carefully rinsed and then dried by using a nitrogen flow. The substrate was then immersed in an EDC/NHSS aqueous solution for 45 min to activate carboxylate groups, followed by a goat anti-human IgG (660 nM) solution at 37 °C with continual rocking for 2 hours. The BSA aqueous solution (1 wt%) was also introduced to avoid nonspecific absorption by incubation for 2 hours at 37 °C. Finally, the Au substrate with immobilized antibody was prepared.

Control experiments were performed to verify the immune function of the nanomaterials. First, the gold substrate was directly immersed into the prepared ZnS:Eu probe solution for 2 h as the blank test. Second, a specified concentration of human IgG molecules (1 μM) was added to the probe solution and rocked for 2 h for analyte capturing. After being centrifuged and redispersed, the solution containing the probes/analyte complexes was prepared. The Au substrate with immobilized antibody was immersed in the solution for 2 h at 37 °C with gentle shaking. Then, the substrate was carefully rinsed and then dried using a nitrogen flow. To verify the sensitivity, five control experiments were performed. The 800 nM (or 600 nM, 400 nM, 200 nM, 100 nM, 10 nM) human IgG was added to the probe solution with all other experimental conditions being the same as those in the second experiment. An unspecific control experiment was also carried out to evaluate the selectivity, where the analyte (human IgG) was replaced with 1-μM rabbit IgG.

Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-2200FS microscope operated at 200 kV. Selected area electronic diffraction (SAED) attached to the TEM was used to analyze the crystal structure. The structure was further determined by x-ray diffraction (XRD, Rigaku D/max 2500). The existence of Eu element and the bonding state were confirmed by x-ray photoelectron spectroscopy (XPS, VG ESCALAB LKII) with a binding energy reference of 284.6 eV for a C 1s line. Fluorescence investigations were performed on the samples by using a Shimadzu RF-5301PC fluorescence (FL) spectrometer with an excitation wavelength of 396 nm. Photoluminescence (PL) investigations of the sample were carried out with a Horiba LabRam HR evolution spectrophotometer excited with a 532-nm laser. For temperature-dependent photoluminescence, the temperature variation range is from 300 K to 100 K.

The morphology and the structure of the synthesized sample are investigated by TEM and SAED measurements. Figure 2(a) shows the high resolution TEM (HRTEM) image of the formed ZnS:Eu nanoparticles with a doping concentration of 4%. The inset in Fig. 2(a) shows a large field TEM image. Clear lattice fringes of the sample are observed. The crystal structure of the ZnS:Eu nanoparticles is determined by using an SAED as shown in Fig. 2(b).

Fig. 2.

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	Fig. 2. (a) HRTEM image, with inset showing TEM image of a larger area, and (b) SAED pattern.

The interplanar distances calculated by using the three diffraction rings are 3.10 Å, 1.90 Å, and 1.57 Å, which coincide with those of the (111), (220), and (311) planes of cubic zinc-blende ZnS, respectively. The results of XRD measurement further confirms the crystal structure (Fig. 3). No other diffraction peaks are observed, indicating no obvious impurities. The positions of the (111) peaks of undoped ZnS and ZnS:Eu (1%–4%) nanocrystals are located at 28.77°, 28.56°, 28.44°, 28.23°, 28.03°, respectively. When Zn²⁺ is replaced by Eu³⁺, lattice expansion of ZnS can be caused, which leads to the shift toward the lower degree direction.^[17] The XPS measurement is performed to confirm the existence of Eu element and the bonding states in ZnS:Eu nanomaterials. As shown in Fig. 4, the characteristic peak at 1135.7 eV is attributed to the core level of Eu 3d 5/2, which indicates that the Eu ions are trivalent.^[18]

Fig. 3.

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	Fig. 3. (color online) XRD pattern of the ZnS:Eu nanomaterials with different doping concentrations.

Fig. 4.

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	Fig. 4. (color online) XPS spectrum of ZnS:Eu (4%): (a) XPS survey spectrum; (b) Eu 3d XPS spectrum.

The influence of Eu³⁺ doping concentration on the luminescence intensity is discussed in this paper. Figure 5 shows the emission spectra of samples all diluted to the same extent with an excitation wavelength of 396 nm. The doping concentration is varied from 1% to 5%. Three emission peaks located at 594 nm, 617 nm, and 714 nm are clearly observed. The weak emission peak, which is located at 594 nm, comes from the emission of the ⁵D₀ → ⁷F₁ transition in Eu³⁺. The strong emission peak at 617 nm is attributed to the ⁵D₀ → ⁷F₂ transition, and the other emission around 714 nm arises from the ⁵D₀ → ⁷F₄ transition.^[19] The intensity ratio between transitions of ⁵D₀ → ⁷F₂ and ⁵D₀ → ⁷F₁ is called the asymmetry ratio, and is used to effectively detect the symmetry of the Eu³⁺ ion crystal lattice through using the spectrum.^[20] The luminescence intensity of the electric dipole transition at 617 nm (⁵D₀ → ⁷F₂) is higher than that of the magnetic dipole transition located in 594 nm (⁵D₀ → ⁷F₁), which can be explained by Eu³⁺ occupying the lattice position off the inversion center.^[21] The increasing of the doping concentration gradually increases the intensity of emission peak, until it reaches up to 4%. When the doping concentration increases to 5%, the fluorescence intensity decreases due to the concentration quenching effect.^[22] The inset of Fig. 5 shows the relationship between the fluorescence intensity and the doping concentration. Whereas the doping concentration has no influence on the peak wavelength of the emission, indicating that the ZnS:Eu sample with any doping concentration possesses characteristic fluorescence which can provide uniform detection result in the following immunoassays.

Fig. 5.

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	Fig. 5. (color online) Fluorescence emission spectra of liquid samples containing ZnS:Eu nanomaterials with different doping concentrations. The inset shows the relationship between the fluorescence intensity and the doping concentration.

Except for the doping concentration, the influence of temperature on the fluorescence is also investigated. Considering the fact that biomolecular may lose activities under high temperature, the temperature range in PL measurement is from 300 K to 100 K. A 532-nm laser is chosen as the excited light to obtain more detailed information about the emissions from Eu³⁺ ion.^[23] With respect to the fluorescence excited by a xenon lamp (as shown in Fig. 5), more ⁵D₀ → ⁷F_J (J = 0, 1, 2, 3, 4) transitions can be observed (see Fig. 6). It is obvious that there is a series of emission sites in Fig. 6, which are the characteristic emissions of Eu³⁺ ion,^[18] corresponding to the ⁵D₀ → ⁷F₀, ⁵D₀ → ⁷F₁, ⁵D₀ → ⁷F₂, ⁵D₀ → ⁷F₃, ⁵D₀ → ⁷F₄ transitions, respectively. As the temperature decreases, the intensity of light emission gradually decreases, which may result from the surface effect and the local environment changes of Eu³⁺ in the surface layers.^[24] However, the peak positions of the luminescence do not change, indicating that the wavelength of the fluorescence is independent of temperature. This will be essential to the distinguishing of the origin of the signal in photoluminescence measurements (the signal is derived from the background or the biolabels), especially for ultralow concentration detections.

Fig. 6.

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	Fig. 6. (color online) Photoluminescence spectra of ZnS:Eu varying with temperature for an excitation wavelength of 532 nm.

The principle used for this assay is similar to a sandwiched enzyme-linked immunosorbent assay (ELISA). Compared with the ELISA, the step of adding an enzymatic substrate is omitted, and the enzyme is replaced by ZnS:Eu nanocrystal, which can provide simpler and more robust assays. The 4% doped ZnS:Eu nanomaterial presents the optimal fluorescence and is chosen as the biolabel to perform immunoassays. A 532-nm laser is used as an excitation source. Figure 7 shows the immune detection results. For sandwiched structures with 1-μM analyte, the characteristic emissions of Eu³⁺ at 714 nm, 617 nm, 594 nm, and 580 nm can be observed. For immune detections, blank and unspecific group experiments can avoid the false positive signal. No analyte is added in the blank control group, and human IgG is replaced by 1-μM rabbit IgG in the unspecific group. The luminescence signal of the ZnS:Eu is observed in neither of the control groups (see Fig. 7), indicating good specificity of the immunoassay. To evaluate the performance of the current assay, the concentration of the analyte decreases from 800 nM to 10 nM. Statistical analysis based on repeated immunoassay indicates that the intensity of the fluorescence gradually decreases with analyte concentration decreasing [Fig. 7(b)]. The regression curve equation is “Y = 17.2226 + 0.0584X”, and correlation coefficient is 0.9921. The linear range is 10 nM–800 nM. The detection limit is worked out by the “3σ/S”, where S is the slope of the curve equation, and σ represents the standard deviation for the blank sample. The calculated detection limit is 9.6 nM. The sensitivity of the current stage ZnS:Eu based assay is similar to that of commercially available ELISA, and the promising prospects can be expected due to the advantages of the current assay.

Fig. 7.

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	Fig. 7. (color online) (a) Photoluminescence spectra of the sandwich structures with different concentrations of the analyte. (b) Plot of photoluminescence intensity at 617 nm versus analyte concentration, with error bars indicating standard deviation.